The investigation of the secondary structure propensities and free-energy landscapes of peptide ligands by replica exchange molecular dynamics simulations

The conformational states of two peptide sequences that bind to staphylococcal enterotoxin B are sampled by replica exchange molecular dynamic (REMD) simulations in explicit water. REMD simulations were treated with 52 replicas in the range of 280–501 K for both peptides. The conformational ensembles of both peptides are dominated by random coil, bend and turn structures with a small amount of helical structures for each temperature. In addition, while an insignificant presence of β-bridge structures were observed for both peptides, the β-sheet structure was observed only for peptide 3. The results obtained from simulations at 300 K are consistent with the experimental results obtained from circular dichroism spectroscopy. From the analysis of REMD results, we also calculated hydrophobic and hydrophilic solvent accessible surface areas for both peptides, and it was observed that the hydrophobic segments of the peptides tend to form bend or turn structures. Moreover, the free-energy landscapes of both peptides were obtained by principal component analysis to understand how the secondary structural properties change according to their complex space. From the free-energy analysis, we have found several minima for both peptides at decreased temperature. For these obvious minima of both peptides, it was observed that the random coil, bend and turn structures are still dominant and the helix, β-bridge or β-sheet structures can appear or disappear with respect to minima. On the other hand, when we compare the results of REMD with conventional MD simulations for these peptides, the configurations of peptide 3 might be trapped in energy minima during the conventional MD simulations. Hence, it can be said that the REMD simulations have provided a sufficiently high sampling efficiency.     

N-Heterocyclic carbene gold(I) and silver(I) adducts of [Pt2(μ-S)2(PPh3)4]

Reaction of the metalloligand [Pt₂(μ-S)₂(PPh₃)₄] with the N-heterocyclic carbene (NHC) complexes IPrAuCl, IMesAuCl and IMesAgCl in methanol gave the first examples of metal adducts of [Pt₂(μ-S)₂(PPh₃)₄] that contain NHC ligands, namely [Pt₂(μ-S)₂(PPh₃)₄AuL]⁺ (L = IPr, IMes) and [Pt₂(μ-S)₂(PPh₃)₄AgIMes]⁺. The complexes were isolated as hexafluorophosphate salts. Reaction of [Pt₂(μ-S)₂(PPh₃)₄] with excess IPrAuCl in refluxing methanol yielded only the mono-adduct, in contrast to the behaviour with the gold(I) phosphine complex Ph₃PAuCl, which undergoes double addition giving [Pt₂(μ-SAuPPh₃)₂(PPh₃)₄]²⁺. The X-ray structure of [Pt₂(μ-S)₂(PPh₃)₄AuIPr]PF₆ was determined and reveals that the ‘free’ sulfide is substantially sterically protected by the IPr ligand, accounting for the low reactivity towards addition of a second AgIPr⁺ moiety.     

Copper(II) complexes with monocarboxylate ligands bearing different substituent groups: Synthesis and spectroscopic studies

In our continuing efforts to explore the effects of substituent groups of ligands in the formation of supramolecular coordination structures, seven new Cu^II complexes formulated as [Cu₂(L¹)₄(DMF)₂] (1), {[Cu₂(L¹)₄(Hmta)](H₂O)_0.75}_∞ (2), [Cu₂(L²)₄(2,2′-bipy)₂] (3), [Cu₂(L³)₄(H₂O)₂] (4), [Cu₂(L³)₄(Hmta)]_∞ (5), [Cu₂(L³)₄(Dabco)]_∞ (6) and [Cu₂(L³)₄(Pz)]_∞ (7) with three monocarboxylate ligands bearing different substituent groups HL¹-HL³ (HL¹ = phenanthrene-9-carboxylic acid, HL² = 2-phenylquinoline-4-carboxylic acid, HL³ = adamantane-1-carboxylic acid, Hmta = hexamethylenetetramine, 2,2′-bipy = 2,2′-bipyridine, Dabco = 1,4-diazabicyclo[2.2.2] octane and Pz = pyrazine), have been prepared and characterized by X-ray diffraction. In 1, 2 and 4-7, each Cu^II ion is octahedrally coordinated, and carboxylate acid acts as a syn-syn bridging bidentate ligand. While each Cu^II ion in 3 is penta-coordinated in a distorted square-pyramidal geometry. 1 and 4 both show a dinuclear paddle-wheel block, while 2, 5, 6 and 7 all exhibit an alternated 1D chain structure between dinuclear paddle-wheel units of the tetracarboxylate type Cu₂-(RCO₂)₄ and the bridging auxiliary ligands Hmta, Dabco and Pz. Furthermore, 3 has a carboxylic unidentate and μ_1,1-oxo bridging dinuclear structure with the chelating auxiliary ligand 2,2′-bipy. Moreover, complexes 1-6 were characterized by electron paramagnetic resonance (EPR) spectroscopy.     

Separation of hexahistidine fusion proteins with immobilized metal ion affinity chromatographic (IMAC) sorbents derived from MN+‐tacn and its derivatives

The capabilities of a new class of immobilized (im) metal ion chelate complexes (IMCCs), derived from 1,4,7‐triazacyclononane (tacn), bis(1,4,7‐triazacyclononyl) ethane (dtne) and bis(1,4,7‐triazacyclononyl)propane (dtnp) complexed with the borderline metal ions Cu²⁺, Ni²⁺, Zn²⁺, Mn²⁺, Co²⁺, and Cr³⁺, for the purification of proteins have been investigated. In particular, the binding behavior of a model protein, the C‐terminal hexahistidine tagged recombinant fusion protein Schistosoma japonicum glutathione S‐transferase‐Saccharomyces cerevisiae mitochondrial ATP synthase δ‐subunit (GST‐δATPase‐His₆), with these new immobilized metal ion affinity chromatographic (IMAC) sorbents was compared to the properties of a conventional sorbent, derived from immobilized Ni(II)‐nitrilotriacetic acid (im‐Ni²⁺‐NTA). Investigations using the recombinant GST‐δATPase‐His₆ and recombinant S. japonicum glutathione S‐transferase (GST) lacking a hexahistidine tag have confirmed that the C‐terminal tag hexahistidine residues were required for the binding process to occur with these IMAC systems. The results also confirm that recombinant fusion proteins such as GST‐δATPase‐His₆ can be isolated in high purity with these IMAC systems. Moreover, these new macrocyclic systems manifest different selectivity features as a function of pH or ionic strength when compared to the conventional, unconstrained iminodiacetic acid (IDA) or NTA chelating ligands, complexed with borderline metal ions such as Cu²⁺ or Ni²⁺, as IMAC systems. Biotechnol. Bioeng. 2009;103: 747–756. © 2009 Wiley Periodicals, Inc.     

核酸适配体及其在病原微生物学中的应用

核酸适配体指利用指数富集配体系统进化技术筛选出的寡聚核苷酸片段,它可以特异性地识别靶标并与之结合,已经广泛应用于基础研究、临床诊断、纳米技术等。以下综述了适配体在微生物学方面的应用。     

Molecular evidence and clinical importance of β‐arrestins expression in patients with acromegaly

β‐arrestins seem to have a role in endocytosis and desensitization of somatostatin receptor subtype 2 (sst2) and could be associated with the responsiveness to somatostatin receptor ligands (SRL) in patients with acromegaly. To investigate the in vivo correlation between β‐arrestins 1 and 2 with sst2, sst5 and dopamine receptor subtype 2 (D2) expressions, and the association of β‐arrestins with response to first‐generation SRL and invasiveness in somatotropinomas. β‐arrestins 1 and 2, sst2, sst5 and D2 mRNA expressions were evaluated by quantitative real‐time RT‐PCR on tumoral tissue of 96 patients. Moreover, sst2 and sst5 protein expressions were also evaluated in 40 somatotropinomas by immunohistochemistry. Response to SRL, defined as GH <1 μg/l and normal IGF‐I levels, was assessed in 40 patients. The Knosp‐Steiner criteria were used to define invasiveness. Median β‐arrestin 1, β‐arrestin 2, sst2, sst5 and D2 mRNA copy numbers were 478; 9375; 731; 156 ; and 3989, respectively. There was a positive correlation between β‐arrestins 1 and 2 (R = 0.444, P < 0.001). However, no correlation between β‐arrestins and sst2, sst5 (mRNA and protein levels) or D2 was found. No association was found between β‐arrestins expression and SRL responsiveness or tumour invasiveness. Although previous data suggest a putative correlation between β‐arrestins and sst2, our data clearly indicated that no association existed between β‐arrestins and sst2, sst5 or D2 expression, nor with response to SRL or tumour invasiveness. Therefore, further studies are required to clarify whether β‐arrestins have a role in the response to treatment with SRL in acromegaly.     

Purification and characterization of a benzodiazepine-like substance from mammalian brain

An endogenous brain ligand which competes with [³H]-flunitrazepam for the binding to benzodiazepine receptor has been isolated and purified to homogeneity. The purification procedures involve the extraction of the endogenous ligand by homogenizing the brain tissue in water containing various protease inhibitors followed by filtration through a PM 10 membrane (exclusion limit: 10,000-dalton), column chromatographies on Sephadex G-50, Bio-Rad P2 and a series of C₁₈ reverse phase HPLC columns. The purified endogenous ligand was eluted as a single and symmetrical peak monitored at either 220 or 280 nm. Furthermore, the ligand activity coincided with the absorption peak. The purified endogenous ligand is thermostable, insensitive to various peptidases and proteolytic enzymes, resistant to DNAse, RNAse, and carbohydrate enzyme e.g. neuraminidase (EC 3.2.1.18) and acid treatment. It has a major absorption peak at 220 nm and a minor one at 313 nm. The endogenous ligand appears to be quite specific since it only inhibits the binding of ligand to the central type benzodiazepine receptor but not to other receptors, e.g. peripheral type benzodiazepine receptor, ₁-adrenoceptor, ₂-adrenoceptor, -adrenoceptor and muscarinic cholinergic receptor. Furthermore, the inhibition of the receptor binding by the endogenous ligand is enhanced by GABA suggesting that the endogenous ligand is a benzodiazepine receptor agonist. The structure of the endogenous ligand is unknown.Special issue dedicated to Dr. Elling Kvamme     

Transition metal complexes based on pyridine ligand containing both bis(2-pyridyl) and 1,3-dithiole-2-ylidene units: Syntheses, structures, and magnetic studies

Two new mononuclear spin-crossover iron(II) complexes, [FeL₂(NCS)₂] · H₂O (1) and [FeL₂(NCSe)₂] (2), have been synthesized from the reaction of the versatile ligand 4,5-bis(2-cyanoethylthio)-2-bis(2-pyridyl)methylene-1,3-dithiole (L), Fe(ClO₄)₂, and KNCX (X = S/Se). Reactions of L with Cu^II or Co^II salts afford one mononuclear complex [CuL(hfac)₂] · CH₃OH (hfac = hexafluoroacetylacetonate) (3), one dinuclear complex [(CuLCl)₂(μ-Cl)₂] · CH₃OH (4), and two 1D chain species, [CuL₂]_n(BF₄)_2n (5) and [CoL₂]_n(ClO₄)_2n · 2nCH₂Cl₂ (6). The crystal structures of complexes 1 and 3-6 have been determined by X-ray crystallography. Short intermolecular S?S contacts between neighboring 1D arrays are observed in 5 and 6, which lead to the formation of the 2D structure. The magnetic properties are studied, and antiferromagnetic couplings between the Cu^II centers across the chloride bridges have been found in 4 (J = 2.04 cm^-1). Spin-crossover behaviors between high and low spin states are observed at T_1/2 = 80 K for 1 and T_1/2 = 300 K for 2, respectively.     

5S rRNA-assisted DnaK refolding

Although accumulating evidence has revealed that most proteins can fold without the assistance of molecular chaperones, little attention has been paid to other types of chaperoning macromolecules. A variety of proteins interact with diverse RNA molecules in vivo, suggesting a potential role of RNAs for folding of their interacting proteins. Here we show that the in vitro refolding of a representative molecular chaperone, DnaK, an Escherichia coli homolog of Hsp70, could be assisted by its interacting 5S rRNA. The folding enhancement occurred in RNA concentration and its size dependent manner whereas neither the RNA with the reverse sequence of 5S rRNA nor the RNase pretreated 5S rRNA stimulated the folding in vitro. Based on our results, we propose that 5S rRNA could exert the chaperoning activity on DnaK during the folding process. The results suggest an interesting possibility that the folding of RNA-interacting proteins could be assisted by their cognate RNA ligands.     

Dimeric argininamide-type neuropeptide Y receptor antagonists: Chiral discrimination between Y1 and Y4 receptors

The structurally related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are endogenous agonists of the NPY receptor (YR) family, which in humans comprises four functionally expressed subtypes, designated Y₁R, Y₂R, Y₄R and Y₅R. Nonpeptide antagonists with high affinity and selectivity have been described for the Y₁R, Y₂R and Y₅R, but such compounds are still lacking for the Y₄R. In this work, the structures of the high affinity selective (R)-argininamide-type Y₁R antagonists BIBP3226 and BIBO3304 were linked via the guanidine or urea moieties to give homo-dimeric argininamides with linker lengths ranging from 31 to 41 atoms. Interestingly, the twin compounds proved to be by far less selective for the Y₁R than the R-configured monovalent parent compounds. The decrease in selectivity ratio was most pronounced for Y₁R versus Y₄R subtype, resulting in comparable affinities of bivalent ligands for Y₁R and Y₄R (e.g. UR-MK177 ((R,R)-49): K_i = 230 nM (Y₁R) and 290 nM (Y₄R)). With a K_i value of 130 nM and a K_b value of 20 nM, UR-MK188 ((R,R)-51) was superior to all Y₄R antagonists known to date. The S,S-configured optical antipodes of UR-MK177 and UR-MK188 (UR-MEK381 ((S,S)-49) and UR-MEK388 ((S,S)-51)) were synthesized to investigate the stereochemical discrimination by the different receptor subtypes. Whereas preference for R,R-configured argininamides was characteristic of the Y₁R, stereochemical discrimination by the Y₄R was not observed. This may pave the way to selective Y₄R antagonists.